Actobindin, an 88-amino acid protein from Acanthamoeba castellanii that is a potent inhibitor of an early step in actin polymerization, forms a ternary complex with 2 molecules of G-actin. The interface between actobindin and actin in the complex has been defined by cross-linking studies using the zero length crosslinker EDC. Lysines 16 and 52 (residues at the beginning of the two internal repeat of 33-34 amino acids) of actobindin were cross-linked to one of the acid amino acids at actin residues 1, 2 or 3. Lysine 16 of actobindin was also crosslinked to glutamate 100 of actin. Actobindin has also been shown to bind to covalently cross-linked actin dimers with a higher affinity than to 2 actin monomers in a manner consistent with the previously determined affinities for each of the 2 monomers. The covalently cross-linked actin dimers may be models of the small oligomer(s) with which actobindin may interact physiologically.